Our long-term goal is to discover broad-spectrum antibiotics acting against potential biowarfare agents. The goal of this exploratory project is to develop a comprehensive approach to bypass the existing obstacles to drug discovery, which will include delivering active compounds into the pathogen, identifying new classes of antimicrobials, and efficient evaluation of toxicity/efficacy. Previous research in the Lewis laboratory showed that plants synthesize inhibitors of multi-drug resistant efflux pumps that can facilitate delivery of antimicrobials into microbial pathogens. Independently, the Ausubel laboratory developed a pathogenesis model that involves the killing of the nematode worm Caenorhabditis elegans by human microbial pathogens. The nematode can therefore be used as an animal model for primary screening of antimicrobials. This proposal describes experiments designed to merge the complementary technologies developed in these two laboratories to produce a novel approach to antimicrobial drug discovery. The Specific Aims are: 1. High throughput whole-animal screen for novel antimicrobials will be developed using C. elegans infected with a variety of NIAID group A and B agents. These will include diarrheagenic E. coli, S. enterica, and a model gram-positive pathogen E. faecalis. We will also establish whether C. elegans is infected with B. anthracis and F. tularensis. The rationale of the antimicrobial assay is to monitor curing of worms infected with human pathogens, by test compounds. A liquid assay using GFP-labeled C. elegans will be developed into a high-throughput automated assay. 2. Screening for MDR inhibitors and new antimicrobials will be performed in vitro, and in vivo with infected C. elegans. A commercial synthetic compound library, and the NCI collection of extracts will be screened. Comparison of the in vitro and in vivo screens will identify possible compounds that only act in vivo (prodrugs, compounds targeting virulence or other components necessary for in vivo survival, and stimulators of innate immunity). Preliminary results show that the NCI library has hits for both direct and MDR inhibitory activity against all pathogens tested. We will focus on obtaining novel antimicrobials and MDR inhibitors acting against gram-negative pathogens. A combination of such an MDR inhibitor with an antimicrobial compound will produce a broad-spectrum antibiotic. We plan to screen 30,000 compounds/extracts in this pilot study. 3. We will purify and identify antimicrobial compounds. Active extracts will be used to isolate a pure substance. A sufficiently pure compound will be used to determine MIC with the pathogen panel, and those with high potency will be studied further. Determination of chemical structure will be performed by a combination of MS and NMR methods.